Interaction of hepatitis B virus X protein with damaged DNA-binding protein p127: Structural analysis and identification of antagonists

The hepatitis B virus X protein is a multifunctional protein that is essential for natural infection and has also been implicated in liver cancer development. Previous studies have identified the DDB1 subunit of the damaged-DNA binding complex as a critical partner of X protein in the infection process, X-mediated cytotoxicity and stability of the viral protein. Here, we investigated the structural and functional constraints of X-DDB1 interaction using various mutational analyses. Our data show that the interaction interface of X with DDB1 is confined to a 15-residue epitope. All substitutions responsible for loss of binding mapped to this core-binding domain. In contrast, a marked increase in affinity for DDB1 resulted from substitutions at clustered positions lying close to the DDB1-binding epitope and correlated with loss of apoptotic potential. Selection of mutations in DDB1 that partially rescue the binding defect of an X mutant gave further insight into the contacts established between the two proteins. Importantly, both the core-binding domain of X and the gain-of-affinity X mutants inhibited DDB1-mediated stabilization of wild-type X protein. These X protein derivatives thus provide the basis for the development of therapeutic agents that antagonize X function through competitive inhibition of X-DDB1 interaction.     

Evaluation of mono or mixed cultures of lactic acid bacteria in type II sourdough system

The aim of this study was to evaluate the use of mono and mixed lactic acid bacteria (LAB) cultures to determine suitable LAB combinations for a type II sourdough system. In this context, previously isolated sourdough LAB strains with antimicrobial activity, which included Lactobacillus plantarum PFC22, Lactobacillus brevis PFC31, Pediococcus acidilactici PFC38, and Lactobacillus sanfranciscensis PFC80, were used as mono or mixed culture combinations in a fermentation system to produce type II sourdough, and subsequently in bread dough production. Compared to the monoculture fermentation of dough, the use of mixed cultures shortened the adaptation period by half. In addition, the use of mixed cultures ensured higher microbial viability, and enhanced the fruity flavor during bread dough production. It was determined that the combination of L. plantarum PFC22 + P. acidilactici PFC38 + L. sanfranciscensis PFC80 is a promising culture mixture that can be used in the production of type II sourdough systems, and that may also contribute to an increase in metabolic activity during bread production process.     

Sodium selenide toxicity is mediated by O2-dependent DNA breaks

Hydrogen selenide is a recurrent metabolite of selenium compounds. However, few experiments studied the direct link between this toxic agent and cell death. To address this question, we first screened a systematic collection of Saccharomyces cerevisiae haploid knockout strains for sensitivity to sodium selenide, a donor for hydrogen selenide (H(2)Se/HSe(-/)Se(2-)). Among the genes whose deletion caused hypersensitivity, homologous recombination and DNA damage checkpoint genes were over-represented, suggesting that DNA double-strand breaks are a dominant cause of hydrogen selenide toxicity. Consistent with this hypothesis, treatment of S. cerevisiae cells with sodium selenide triggered G2/M checkpoint activation and induced in vivo chromosome fragmentation. In vitro, sodium selenide directly induced DNA phosphodiester-bond breaks via an O(2)-dependent reaction. The reaction was inhibited by mannitol, a hydroxyl radical quencher, but not by superoxide dismutase or catalase, strongly suggesting the involvement of hydroxyl radicals and ruling out participations of superoxide anions or hydrogen peroxide. The (?)OH signature could indeed be detected by electron spin resonance upon exposure of a solution of sodium selenide to O(2). Finally we showed that, in vivo, toxicity strictly depended on the presence of O(2). Therefore, by combining genome-wide and biochemical approaches, we demonstrated that, in yeast cells, hydrogen selenide induces toxic DNA breaks through an O(2)-dependent radical-based mechanism.     

A selective sweep driven by pyrimethamine treatment in southeast asian malaria parasites

Malaria parasites (Plasmodium falciparum) provide an excellent system in which to study the genomic effects of strong selection in a recombining eukaryote because the rapid spread of resistance to multiple drugs during the last the past 50 years has been well documented, the full genome sequence and a microsatellite map are now available, and haplotype data can be easily generated. We examined microsatellite variation around the dihydrofolate reductase (dhfr) gene on chromosome 4 of P. falciparum. Point mutations in dhfr are known to be responsible for resistance to the antimalarial drug pyrimethamine, and resistance to this drug has spread rapidly in Southeast (SE) Asia after its introduction in 1970s. We genotyped 33 microsatellite markers distributed across chromosome 4 in 61 parasites from a location on the Thailand/Myanmar border. We observed minimal microsatellite length variation in a 12-kb (0.7-cM) region flanking the dhfr gene and diminished variation for approximately 100 kb (6 cM), indicative of a single origin of resistant alleles. Furthermore, we found the same or similar microsatellite haplotypes flanked resistant dhfr alleles sampled from 11 parasite populations in five SE Asian countries indicating recent invasion of a single lineage of resistant dhfr alleles in locations 2000 km apart. Three features of these data are of especially interest. (1). Pyrimethamine resistance is generally assumed to have evolved multiple times because the genetic basis is simple and resistance can be selected easily in the laboratory. Yet our data clearly indicate a single origin of resistant dhfr alleles sampled over a large region of SE Asia. (2). The wide valley ( approximately 6 cM) of reduced variation around dhfr provides "proof-of-principle" that genome-wide association may be an effective way to locate genes under strong recent selection. (3). The width of the selective valley is consistent with predictions based on independent measures of recombination, mutation, and selection intensity, suggesting that we have reasonable estimates of these parameters. We conclude that scanning the malaria parasite genome for evidence of recent selection may prove an extremely effective way to locate genes underlying recently evolved traits such as drug resistance, as well as providing an opportunity to study the dynamics of selective events that have occurred recently or are currently in progress.     

DNA oligonucleotides with A, T, G or C opposite an abasic site: structure and dynamics

Abasic sites are common DNA lesions resulting from spontaneous depurination and excision of damaged nucleobases by DNA repair enzymes. However, the influence of the local sequence context on the structure of the abasic site and ultimately, its recognition and repair, remains elusive. In the present study, duplex DNAs with three different bases (G, C or T) opposite an abasic site have been synthesized in the same sequence context (5′-CCA AAG₆ XA₈C CGG G-3′, where X denotes the abasic site) and characterized by 2D NMR spectroscopy. Studies on a duplex DNA with an A opposite the abasic site in the same sequence has recently been reported [Chen,J., Dupradeau,F.-Y., Case,D.A., Turner,C.J. and Stubbe,J. (2007) Nuclear magnetic resonance structural studies and molecular modeling of duplex DNA containing normal and 4′-oxidized abasic sites. Biochemistry, 46, 3096–3107]. Molecular modeling based on NMR-derived distance and dihedral angle restraints and molecular dynamics calculations have been applied to determine structural models and conformational flexibility of each duplex. The results indicate that all four duplexes adopt an overall B-form conformation with each unpaired base stacked between adjacent bases intrahelically. The conformation around the abasic site is more perturbed when the base opposite to the lesion is a pyrimidine (C or T) than a purine (G or A). In both the former cases, the neighboring base pairs (G6-C21 and A8-T19) are closer to each other than those in B-form DNA. Molecular dynamics simulations reveal that transient H-bond interactions between the unpaired pyrimidine (C20 or T20) and the base 3′ to the abasic site play an important role in perturbing the local conformation. These results provide structural insight into the dynamics of abasic sites that are intrinsically modulated by the bases opposite the abasic site.     

Fanconi DNA repair pathway is required for survival and long-term maintenance of neural progenitors

Although brain development abnormalities and brain cancer predisposition have been reported in some Fanconi patients, the possible role of Fanconi DNA repair pathway during neurogenesis is unclear. We thus addressed the role of fanca and fancg, which are involved in the activation of Fanconi pathway, in neural stem and progenitor cells during brain development and adult neurogenesis. Fanca(-/-) and fancg(-/-) mice presented with microcephalies and a decreased neuronal production in developing cortex and adult brain. Apoptosis of embryonic neural progenitors, but not that of postmitotic neurons, was increased in the neocortex of fanca(-/-) and fancg(-/-) mice and was correlated with chromosomal instability. In adult Fanconi mice, we showed a reduced proliferation of neural progenitor cells related to apoptosis and accentuated neural stem cells exhaustion with ageing. In addition, embryonic and adult Fanconi neural stem cells showed a reduced capacity to self-renew in vitro. Our study demonstrates a critical role for Fanconi pathway in neural stem and progenitor cells during developmental and adult neurogenesis.     

Formation of eicosanoids, E2/D2 isoprostanes, and docosanoids following decapitation-induced ischemia, measured in high-energy-microwaved rat brain

Inflammatory lipid mediators derived from arachidonic acid (AA) and docosahexaenoic acid (DHA) modify the pathophysiology of brain ischemia. The goal of this work was to investigate the formation of eicosanoids and docosanoids generated from AA and DHA, respectively, during no-flow cerebral ischemia. Rats were subjected to head-focused microwave irradiation 5 min following decapitation (complete ischemia) or prior to decapitation (controls). Brain lipids were extracted and analyzed by reverse-phase liquid chromatography-tandem mass spectrometry. After complete ischemia, brain AA, DHA, and docosapentaenoic acid concentrations increased 18-, 5- and 4-fold compared with controls, respectively. Prostaglandin E(2) (PGE(2)) and PGD(2) could not be detected in control microwaved rat brain, suggesting little endogenous PGE(2)/D(2) production in the brain in the absence of experimental manipulation. Concentrations of thromboxane B(2), E(2)/D(2)-isoprostanes, 5-hydroxyeicosatetraenoic acid (5-HETE), 5-oxo-eicosatetraenoic acid, and 12-HETE were significantly elevated in ischemic brains. In addition, DHA products such as mono-, di- and trihydroxy-DHA were detected in control and ischemic brains. Monohydroxy-DHA, identified as 17-hydroxy-DHA and thought to be the immediate precursor of neuroprotectin D(1), was 6.5-fold higher in ischemic than in control brain. The present study demonstrated increased formation of eicosanoids, E(2)/D(2)-IsoPs, and docosanoids following cerebral ischemia. A balance of these lipid mediators may mediate immediate events of ischemic injury and recovery.     

Microhabitat amelioration and reduced competition among understorey plants as drivers of facilitation across environmental gradients: Towards a unifying framework

Studies of facilitative interactions as drivers of plant richness along environmental gradients often assume the existence of an overarching stress gradient that equally affects the performance of all the species in a given community. However, co-existing species differ in their ecophysiological adaptations, and do not experience the same stress level under particular environmental conditions. Moreover, these studies assume a unimodal relationship between richness and biomass, which is not as general as previously thought. We ignored these assumptions to assess changes in plant–plant interactions and their effect on local species richness across environmental gradients in semi-arid areas of Spain and Australia. We aimed to understand the relative importance of direct (microhabitat amelioration) and indirect (changes in the competitive relationships among the understorey species: niche segregation, competitive exclusion or intransitivity) mechanisms that might underlie the effects of nurse plants on local species richness. By jointly studying these direct and indirect mechanisms using a unifying framework, we found that nurse plants (trees, shrubs and tussock grasses) increased local richness not only by expanding the niche of neighbouring species but also by increasing niche segregation among them, though the latter was not important in all cases. The outcome of the competition-facilitation continuum varied depending on the study area, likely because the different types of stress gradient considered. When driven by both rainfall and temperature, or rainfall alone, the community-wide importance of nurse plants remained constant (Spanish sites), or showed a unimodal relationship along the gradient (Australian sites). This study expands our understanding of the relative roles of plant–plant interactions and environmental conditions as drivers of local species richness in semi-arid environments. The results can also be used to refine predictions about the response of plant communities to environmental change, and to clarify the relative importance of biotic interactions as drivers of such responses.     

Chestnut cultivar diversification process in the Iberian Peninsula, Canary Islands, and Azores

This is a large-scale molecular study based on simple sequence repeat (SSR) loci of the diversification process in chestnut cultivars from Portugal and Spain, from the northern Iberian Peninsula to the Canary Islands and the Azores. A total of 593 grafted chestnut trees (Castanea sativa Mill.) were analysed with 10 SSRs: 292 from Portugal and 301 from Spain. Some of the trees studied were more than 300 years old. Accessions were analysed using a model-based Bayesian procedure to assess the geographical structure and to assign individuals to reconstructed populations based on the SSR genotypes. We found 356 different genotypes with a mean value of clonality of 33% owing to grafting. Mutations accounted for 6%, with hybridization being the main diversification process that can explain the great diversity found. Ten main cultivar groups were detected: four in northern Spain, five in the centre of the Iberian Peninsula, and one in southern Spain related to the centre of the Iberian Peninsula. This work demonstrated that cultivar origin and the diversification process was a combination of clonal propagation of selected seedlings, hybridization, and mutations, which allowed high levels of diversity to be maintained with respect to selected clones for fruit production. Furthermore, seedlings and graft sticks facilitated the transport to new destinations in the colonization process, transporting sometimes more than 3000 km if we consider the Azores and the Canary Islands.     

Levels and determinants of inflammatory biomarkers in a Swiss population-based sample (CoLaus study)

Objective

to assess the levels and determinants of interleukin (IL)-1β, IL-6, tumour necrosis factor (TNF)-α and C-reactive protein (CRP) in a healthy Caucasian population.

Methods

population sample of 2884 men and 3201 women aged 35 to 75. IL-1β, IL-6 and TNF-α were assessed by a multiplexed particle-based flow cytometric assay and CRP by an immunometric assay.

Results

Spearman rank correlations between duplicate cytokine measurements (N = 80) ranged between 0.89 and 0.96; intra-class correlation coefficients ranged between 0.94 and 0.97, indicating good reproducibility. Among the 6085 participants, 2289 (37.6%), 451 (7.4%) and 43 (0.7%) had IL-1β, IL-6 and TNF-α levels below detection limits, respectively. Median (interquartile range) for participants with detectable values were 1.17 (0.48–3.90) pg/ml for IL-1β; 1.47 (0.71–3.53) pg/ml for IL-6; 2.89 (1.82–4.53) pg/ml for TNF-α and 1.3 (0.6–2.7) ng/ml for CRP. On multivariate analysis, greater age was the only factor inversely associated with IL-1β levels. Male sex, increased BMI and smoking were associated with greater IL-6 levels, while no relationship was found for age and leisure-time PA. Male sex, greater age, increased BMI and current smoking were associated with greater TNF-α levels, while no relationship was found with leisure-time PA. CRP levels were positively related to age, BMI and smoking, and inversely to male sex and physical activity.

Conclusion

Population-based levels of several cytokines were established. Increased age and BMI, and to a lesser degree sex and smoking, significantly and differentially impact cytokine levels, while leisure-time physical activity has little effect.